人脱细胞羊膜负载大鼠骨髓间充质干细胞构建组织工程膀胱的实验研究
|
摘要
第一部分大鼠骨髓间充质干细胞的分离、培养以及诱导、分化成平滑肌样细胞的实验研究
目的:采用全骨髓培养法结合贴壁法从大鼠骨髓中分离骨髓间充质干细胞,在体外培养、纯化、扩增、传代,应用1%二甲基亚砜(DMSO)和大鼠膀胱组织匀浆上清液诱导分化成平滑肌样细胞,探讨为组织工程膀胱的构建提供种子细胞的可能性。
方法:无菌条件下取大鼠双侧股骨、胫骨和胸骨,用吸取D-Hank’s液的注射器冲出骨髓细胞,置于含10%FBS、100u/ml双抗的L-DMEM培养基中,37℃、5%CO2环境下进行培养,应用全骨髓培养法结合贴壁分离的方法进行纯化。待细胞达95%融合时,用0.125%的胰酶-0.02%的EDTA消化2-3min后进行传代。倒置相差显微镜观察原代及传代后的细胞生长特点。HE染色光镜观察细胞形态特征。扫描电镜(SEM)观察细胞表面的超微结构特征。利用MTT法测定大鼠骨髓间充质干细胞生长曲线,了解细胞生长特性。取第4代大鼠骨髓间充质干细胞用1%二甲亚砜(DMSO)预诱导8小时,然后应用大鼠膀胱组织匀浆上清液诱导7天。倒置相差显微镜观察诱导分化后的大鼠骨髓间充质干细胞形态学的变化并运用免疫细胞化学方法检测特异性蛋白标志物α-平滑肌肌动蛋白的表达。选用未进行诱导的MSCs作为阴性对照组。
结果:原代培养的骨髓细胞成分复杂, 1-2天开始有少量细胞贴壁,形状不规则,有长梭形、多角形等。半量换液后,克隆生长的细胞团块开始迅速增殖,以集落生长为主。7-9天左右细胞可长满培养瓶底,达95%以上融合,主要呈长梭形,其中散在少量折光性强的小圆形细胞。细胞整体排列更趋于规律性,呈漩涡状,辐射状或鱼群样排列。传代后的细胞贴壁及生长更加迅速,2h后部分细胞即开始贴壁,24h内即可完全贴壁,6～7天可铺满培养瓶底。细胞形态更均一,长梭形,整体呈漩涡状,辐射状或鱼群样排列。HE染色示胞体以纺锤形居多,有突起,细胞核大、深染,核仁明显。扫描电镜观察主要为三种形态的细胞:宽大扁平形的细胞为主,长梭形和圆球形细胞占少数。表面可见大量微绒毛样突起,类似树突。P2、P4、P6细胞生长曲线基本相同,分为潜伏期(第1-2天)、对数生长期(第3-5天)和平台期(第6-7天),且几代细胞间生长状况稳定。第4代大鼠骨髓间质干细胞经过1%二甲基亚砜(DMSO)和大鼠膀胱组织匀浆上清液诱导可分化成为梭形平滑肌样细胞,融合后形成峰谷状排列,部分细胞表达平滑肌特异性蛋白标志物α-肌动蛋白。其诱导分化率为(45.6±3.5)%,阴性对照组的分化率为(3.8±0.77)%,二者相比差异有统计学意义(p<0.05)。
结论:1.应用全骨髓培养法结合贴壁分离法可以快速的分离、纯化大鼠骨髓间充质干细胞,并使其在体外迅速得到了增殖,而且性状稳定。
2. P2、P4、P6大鼠骨髓间充质干细胞生长曲线基本相同,细胞增殖快,且几代细胞间生长状况稳定。
3.大鼠骨髓间充质干细胞在体外经膀胱组织匀浆上清液诱导可分化为平滑肌样细胞,可作为膀胱修复的种子细胞进一步研究,从而为后续实验研究打下基础。
第二部分生物支架材料—人脱细胞羊膜的制备
目的:探讨人脱细胞羊膜的制备方法及其作为生物支架材料应用于组织工程膀胱构建的可行性。
方法:取新鲜人羊膜,钝性分离羊膜与绒毛膜组织,去除羊膜基底面残存的绒毛膜和血管组织,采用去污剂洗涤法和酶消化进行脱细胞处理。处理后的材料进行HE染色及电镜检查有无细胞成分残留。以诱导分化的大鼠骨髓间充质干细胞为鉴定细胞接种于96孔板中,分别采用人脱细胞羊膜浸提液及完全培养基进行培养,应用MTT法进行细胞毒性测定。将6-8片处理好的单层脱细胞羊膜按彼此纤维方向垂直的位置重叠,以200W的可见光照射3h,使其交联增厚,增加张力。
结果:制备好的单层人脱细胞羊膜为白色半透明的薄膜,有一定弹性及韧性,厚约0.1mm。光镜观察无细胞成分残留,扫描电镜观察为有胶原蛋白形成的多微孔结构,无细胞残留。细胞毒性测定为Ⅰ级,细胞相容性好。交联后的人脱细胞羊膜厚度0.6-0.8mm,张力大大增加,能耐受缝合和牵拉。
结论:采用去污剂洗涤和酶消化可以对新鲜人羊膜进行脱细胞处理获得脱细胞羊膜。所获支架材料具有一定的弹性、韧性及多微孔结构,为种子细胞的贴附和代谢提供了较好的物质基础。同时该支架材料细胞毒性低、具有良好的组织相容性。交联后的脱细胞羊膜厚度及张力大大增加,能耐受缝合和牵拉,是理想的组织工程用生物支架材料。
第三部分组织工程膀胱的体外构建及大鼠膀胱替代实验
目的:通过采用诱导分化的大鼠骨髓间充质干细胞(MSCs)和人脱细胞羊膜(HAAM)构建组织工程膀胱的实验研究,探讨应用组织工程膀胱进行大鼠膀胱替代修复的可行性。
方法:以诱导分化成功的大鼠骨髓间充质干细胞(MSCs)作为种子细胞,以制备好的人脱细胞羊膜(HAAM)作为生物支架材料,将MSCs静态接种于HAAM表面,进行体外培养5天,获得组织工程膀胱。倒置相差显微镜观察细胞贴附情况。HE染色及扫描电镜(SEM)观察组织工程膀胱情况。将雄性SD大鼠30只随机分为A、B、C 3组,A、B两组分别12只,C组6只,均行半膀胱切除术,然后分别采用负载MSCs的HAAM(A组)、单纯HAAM(B组)以及直接缝合(C组)的方法进行膀胱修补。于术后2、4、8周分别测定膀胱最大容积及解剖取材观察膀胱组织再生情况,8周时行膀胱造影检查。
结果:HAAM与MSCs复合培养第1～2天细胞增殖不明显,第3天时开始有细胞直接贴附于HAAM。随时间延长,贴附的细胞逐渐增多。复合培养第5天,HE染色可见MSCs贴附于羊膜,胞体较大,呈长梭形,细胞形态一致。扫描电镜观察可见细胞散在贴附在材料表面,伸有伪足,并生长入支架材料的孔隙中。大鼠膀胱重建术后2周时A组膀胱的Volmax为1.21±0.60ml,B组为1.12±0.53ml,两组比较无统计学意义(p>0.05),C组为0.75±0.41ml,与A、B两组比较差别有统计学意义(p<0.01)。随时间延长,4周、8周时大鼠的膀胱最大容积略有增加,但A、B两组仍无差别。术后8周时膀胱造影检查可见A、B两组膀胱形态大致正常,而C组膀胱形态明显缩小。大体标本检查: A、B组术后2周膀胱修补吻合区愈合良好,无漏尿;4周时膀胱修补吻合区与正常组织无界限。8周时已经近似正常膀胱,基本为正常组织所替代。膀胱腔内均无结石形成。修复区病理组织学观察:2周时A、B两组支架材料即已降解吸收,修补区大量炎性细胞浸润,内侧可见膀胱移行上皮细胞生长。A组可见平滑肌细胞形成,B组无明显平滑肌细胞。4周时浸润的炎性细胞明显减少,大量毛细血管长入。A组平滑肌细胞增多,B组稀疏可见平滑肌细胞。8周时膀胱的三层结构明显,炎性反应基本消失,A组吻合修补区较厚,平滑肌细胞规则且丰富。接近正常膀胱。B组修补区较薄,平滑肌细胞不规则。而C组仅2周时有轻微炎性反应,4、8周炎性反应消失。膀胱三层结构均正常。
结论:利用MSCs和HAAM采用静态接种培养的方法在体外成功地构建了组织工程膀胱。所构建的组织工程膀胱进行大鼠膀胱替代实验,支架材料降解吸收迅速,平滑肌细胞再生良好,是理想的膀胱替代修补材料。
PartⅠThe studies on separation and cultivation of rat marrow mesenchymal stromal cells (MSCs) and their differentiation into smooth muscle cells by the supernatant of homogenized bladders in vitro.
Objective: To investigate separation, cultivation and purification of murine marrow mesenchymal stromal cells(MSCs) in vitro and its differentiation into smooth muscle cells by the supernatant of homogenized bladder so as to contribute to the seed cells of bladder tissue engineering.
Methods: MSCs were collected from degermed femurs, tibias and sternum of 4 to 6-week-old SD rat by flushing the shaft with buffer (D-Hank’s, PH 7.2) using a syringe with a No. 26 G needle. Cells were cultivated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 100u/mL penicillin-streptomycin). When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The morphologic characteristics of MSCs were studied by HE staining and scanning electron microscope (SEM). The growth curve was tested by MTT assay. The 4th MSCs were cultured in DMEM with 1% DMSO 8 hours before changing the supernatant of homogenized bladders to induce differentiation. Morphology of cells was observed and immunocytochemistry was performed to detect the expression of specificα-SMA.
Results: The components of primarily cultured MSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clones began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. The HE staining result showed that the cell body was fusiform, and some of them had processes. The big and deep staining basophilia nucleus were in the middle of the cell body. Under SEM we could find three kinds of cell morphologies: the most were the large flattened cells and there were a few spindle-shaped and globe cells. On the cells surface we could see a large amount of short and thick microvillus. The growth curve of P2, P4, P6 were quite similar and the cells biological characters kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase. After induction, MSCs demonstrated smooth muscle cell-like morphology and a hill-and-valley growth pattern as well as expressed the specificα-SMA. Its differentiation rate was (45.6±3.5)% vs control group’s(3.8±0.77)% ,there was significant deviation between the two groups.
Conclusion: 1.In this part, a simple new method which was used for the separation, purification and cultivation in vitro of MSC from SD rat bone marrow by total marrow culture associating with adhering to wall has been established. The MSCs could proliferate immediately and keep their biological character stable in vitro.
2. The cells were noted to have a large expansive potential and a fusiform morphology. The growth curves of P2, P4, P6 were quite similar and the cell biological character kept stable.
3. MSCs could be induced into smooth muscle cells by the supernatant of homogenized bladders in vitro, thus providing sufficient amount of seeds cells to bladder tissue engineering.
Part II: Research the characteristic and preparation of human acellular amniotic membrane graft for tissue engineering applications
Objective:To investigate the preparation method of human acellular amniotic membrane(HAAM) and evaluate the feasibility of using HAAM as biomaterial scaffold to construct tissue engineering bladder.
Methods:Human amniotic membranes were decellularized by the method of enzyme digestion and eradicator washing. The human acellular amniotic membranes (HAAM) were then examined by HE staing and electron microscope to confirm no cell elements remained. Murine marrow mesenchymal stromal cells were implanted in 96-hole-plank and cultured by HAAM extract liquid or normal culture medium. The cytotoxcity of HAAM was tested through MTT assay.6-8 pieces of HAAM were overlapped with fiber trend and exposured under 200W light for 3 hours to increase its thickness and tension.
Results:The HAAM was white and translucent, and the thickness was about 0.1mm. It was lubricous and had good elasticity and toughness. There were no cell elements remained under the examination of optical microscope and electron microscope. Many hole structure and remain figure were seen and rich collagen were observed by scanning electron microscope in HAAM. The cytotoxcity score was I, which indicated that HAAM had a good biocompatibility. The thickness of cross-link HAAM was about 0.6-0.8 mm and its tension was increased.
Conclusion:HAAM can be obtained by enzyme digestion and eradicator washing method. This scaffold has good elasticity and toughness, on which the seeds can adhibit and metabolize. The HAAM has good biocompatibility. The cross-link HAAM is thick and tough to endure the suturation and drag. HAAM can be used as ideal biomaterial scaffold of bladder tissue engineering.
Part III: Construction the tissue engineering bladder and replacement experiment for rat
Objective: To investigate the feasibility of bladder repair and replacement by tissue engineering bladder through the construction of murine tissue engineering bladder by marrow mesenchymal stromal cells(MSCs) and human acellular amniotic membrane graft(HAAMG).
Methods: The MSCs of the rats were implanted in HAAM and cultured for about 5 days. Then the tissue engineering bladders were harvested. The cells were observed with phase contrast microscope and the tissue engineering bladders were observed by HE staining and scanning electron microscope.30 rats were divided into 3 groups randomly and marked with A, B and C. There were 12 animals in group A and B, but 6 animals in group C. Hemicystectomies were performed in all 3 groups. The 12 defects of group A were repaired with the tissue engineering bladders, group B with HAAM grafts and the last 6 defects of group C were sutured directly. The rats underwent postoperative assessment of bladder volume and cystography after 2,4,8 weeks. The animal bladders were obtained after that, through which the tissue regeneration was examined.
Results: There was no evident cell proliferation on the first and second day of coculture of HAAM and MSCs. The cells began to stick to HAAM on the third day and the number of adherent cells increased with the time. On the fifth day of coculture, by HE stain, it could be seen that MSCs adhered to amnion. The cells bodies were bigger, fusiform and uniform. Under scanning electron microscope, there were scattered cells adhered to the surface of material. At 2,4,8 weeks after surgery, the bladder volumes of the group A and B were different significantly as compared with that of the group C(p<0.01), but there is no significant difference between the group A and B(p>0.05 ).The Cystography indicated the morphology of the bladder was normal in A and B group at 8 weeks, but became small in C group. The tissue of the anastomoses area grew well after 2 week in A and B group. Portion of HAAM was absorbed after 2 weeks. The tissue structure of the replacement was somewhat the same as that of the normal one after 4,8 weeks. Through the pathological examination, there were lots of inflammatory cells in the replacement tissue in A and B group after 2 weeks, and these inflammatory cells vanished 8 weeks later. The musculature could be observed in A group after 2 weeks and increased along with the time, but little in B group. The epithelial cells could be observed in both A and B group after 2 weeks and increased with the time.
Conclusion: After transplantation with MSCs/HAAM, epithelial cells and smooth muscle cells can regenerated in the graft, and the graft can be absorbed quickly. The tissue engineering bladder constructed by MSCs and HAAM can be used as an ideal biomaterial to replace and repair the bladder。
目的:采用全骨髓培养法结合贴壁法从大鼠骨髓中分离骨髓间充质干细胞,在体外培养、纯化、扩增、传代,应用1%二甲基亚砜(DMSO)和大鼠膀胱组织匀浆上清液诱导分化成平滑肌样细胞,探讨为组织工程膀胱的构建提供种子细胞的可能性。
方法:无菌条件下取大鼠双侧股骨、胫骨和胸骨,用吸取D-Hank’s液的注射器冲出骨髓细胞,置于含10%FBS、100u/ml双抗的L-DMEM培养基中,37℃、5%CO2环境下进行培养,应用全骨髓培养法结合贴壁分离的方法进行纯化。待细胞达95%融合时,用0.125%的胰酶-0.02%的EDTA消化2-3min后进行传代。倒置相差显微镜观察原代及传代后的细胞生长特点。HE染色光镜观察细胞形态特征。扫描电镜(SEM)观察细胞表面的超微结构特征。利用MTT法测定大鼠骨髓间充质干细胞生长曲线,了解细胞生长特性。取第4代大鼠骨髓间充质干细胞用1%二甲亚砜(DMSO)预诱导8小时,然后应用大鼠膀胱组织匀浆上清液诱导7天。倒置相差显微镜观察诱导分化后的大鼠骨髓间充质干细胞形态学的变化并运用免疫细胞化学方法检测特异性蛋白标志物α-平滑肌肌动蛋白的表达。选用未进行诱导的MSCs作为阴性对照组。
结果:原代培养的骨髓细胞成分复杂, 1-2天开始有少量细胞贴壁,形状不规则,有长梭形、多角形等。半量换液后,克隆生长的细胞团块开始迅速增殖,以集落生长为主。7-9天左右细胞可长满培养瓶底,达95%以上融合,主要呈长梭形,其中散在少量折光性强的小圆形细胞。细胞整体排列更趋于规律性,呈漩涡状,辐射状或鱼群样排列。传代后的细胞贴壁及生长更加迅速,2h后部分细胞即开始贴壁,24h内即可完全贴壁,6～7天可铺满培养瓶底。细胞形态更均一,长梭形,整体呈漩涡状,辐射状或鱼群样排列。HE染色示胞体以纺锤形居多,有突起,细胞核大、深染,核仁明显。扫描电镜观察主要为三种形态的细胞:宽大扁平形的细胞为主,长梭形和圆球形细胞占少数。表面可见大量微绒毛样突起,类似树突。P2、P4、P6细胞生长曲线基本相同,分为潜伏期(第1-2天)、对数生长期(第3-5天)和平台期(第6-7天),且几代细胞间生长状况稳定。第4代大鼠骨髓间质干细胞经过1%二甲基亚砜(DMSO)和大鼠膀胱组织匀浆上清液诱导可分化成为梭形平滑肌样细胞,融合后形成峰谷状排列,部分细胞表达平滑肌特异性蛋白标志物α-肌动蛋白。其诱导分化率为(45.6±3.5)%,阴性对照组的分化率为(3.8±0.77)%,二者相比差异有统计学意义(p<0.05)。
结论:1.应用全骨髓培养法结合贴壁分离法可以快速的分离、纯化大鼠骨髓间充质干细胞,并使其在体外迅速得到了增殖,而且性状稳定。
2. P2、P4、P6大鼠骨髓间充质干细胞生长曲线基本相同,细胞增殖快,且几代细胞间生长状况稳定。
3.大鼠骨髓间充质干细胞在体外经膀胱组织匀浆上清液诱导可分化为平滑肌样细胞,可作为膀胱修复的种子细胞进一步研究,从而为后续实验研究打下基础。
第二部分生物支架材料—人脱细胞羊膜的制备
目的:探讨人脱细胞羊膜的制备方法及其作为生物支架材料应用于组织工程膀胱构建的可行性。
方法:取新鲜人羊膜,钝性分离羊膜与绒毛膜组织,去除羊膜基底面残存的绒毛膜和血管组织,采用去污剂洗涤法和酶消化进行脱细胞处理。处理后的材料进行HE染色及电镜检查有无细胞成分残留。以诱导分化的大鼠骨髓间充质干细胞为鉴定细胞接种于96孔板中,分别采用人脱细胞羊膜浸提液及完全培养基进行培养,应用MTT法进行细胞毒性测定。将6-8片处理好的单层脱细胞羊膜按彼此纤维方向垂直的位置重叠,以200W的可见光照射3h,使其交联增厚,增加张力。
结果:制备好的单层人脱细胞羊膜为白色半透明的薄膜,有一定弹性及韧性,厚约0.1mm。光镜观察无细胞成分残留,扫描电镜观察为有胶原蛋白形成的多微孔结构,无细胞残留。细胞毒性测定为Ⅰ级,细胞相容性好。交联后的人脱细胞羊膜厚度0.6-0.8mm,张力大大增加,能耐受缝合和牵拉。
结论:采用去污剂洗涤和酶消化可以对新鲜人羊膜进行脱细胞处理获得脱细胞羊膜。所获支架材料具有一定的弹性、韧性及多微孔结构,为种子细胞的贴附和代谢提供了较好的物质基础。同时该支架材料细胞毒性低、具有良好的组织相容性。交联后的脱细胞羊膜厚度及张力大大增加,能耐受缝合和牵拉,是理想的组织工程用生物支架材料。
第三部分组织工程膀胱的体外构建及大鼠膀胱替代实验
目的:通过采用诱导分化的大鼠骨髓间充质干细胞(MSCs)和人脱细胞羊膜(HAAM)构建组织工程膀胱的实验研究,探讨应用组织工程膀胱进行大鼠膀胱替代修复的可行性。
方法:以诱导分化成功的大鼠骨髓间充质干细胞(MSCs)作为种子细胞,以制备好的人脱细胞羊膜(HAAM)作为生物支架材料,将MSCs静态接种于HAAM表面,进行体外培养5天,获得组织工程膀胱。倒置相差显微镜观察细胞贴附情况。HE染色及扫描电镜(SEM)观察组织工程膀胱情况。将雄性SD大鼠30只随机分为A、B、C 3组,A、B两组分别12只,C组6只,均行半膀胱切除术,然后分别采用负载MSCs的HAAM(A组)、单纯HAAM(B组)以及直接缝合(C组)的方法进行膀胱修补。于术后2、4、8周分别测定膀胱最大容积及解剖取材观察膀胱组织再生情况,8周时行膀胱造影检查。
结果:HAAM与MSCs复合培养第1～2天细胞增殖不明显,第3天时开始有细胞直接贴附于HAAM。随时间延长,贴附的细胞逐渐增多。复合培养第5天,HE染色可见MSCs贴附于羊膜,胞体较大,呈长梭形,细胞形态一致。扫描电镜观察可见细胞散在贴附在材料表面,伸有伪足,并生长入支架材料的孔隙中。大鼠膀胱重建术后2周时A组膀胱的Volmax为1.21±0.60ml,B组为1.12±0.53ml,两组比较无统计学意义(p>0.05),C组为0.75±0.41ml,与A、B两组比较差别有统计学意义(p<0.01)。随时间延长,4周、8周时大鼠的膀胱最大容积略有增加,但A、B两组仍无差别。术后8周时膀胱造影检查可见A、B两组膀胱形态大致正常,而C组膀胱形态明显缩小。大体标本检查: A、B组术后2周膀胱修补吻合区愈合良好,无漏尿;4周时膀胱修补吻合区与正常组织无界限。8周时已经近似正常膀胱,基本为正常组织所替代。膀胱腔内均无结石形成。修复区病理组织学观察:2周时A、B两组支架材料即已降解吸收,修补区大量炎性细胞浸润,内侧可见膀胱移行上皮细胞生长。A组可见平滑肌细胞形成,B组无明显平滑肌细胞。4周时浸润的炎性细胞明显减少,大量毛细血管长入。A组平滑肌细胞增多,B组稀疏可见平滑肌细胞。8周时膀胱的三层结构明显,炎性反应基本消失,A组吻合修补区较厚,平滑肌细胞规则且丰富。接近正常膀胱。B组修补区较薄,平滑肌细胞不规则。而C组仅2周时有轻微炎性反应,4、8周炎性反应消失。膀胱三层结构均正常。
结论:利用MSCs和HAAM采用静态接种培养的方法在体外成功地构建了组织工程膀胱。所构建的组织工程膀胱进行大鼠膀胱替代实验,支架材料降解吸收迅速,平滑肌细胞再生良好,是理想的膀胱替代修补材料。
PartⅠThe studies on separation and cultivation of rat marrow mesenchymal stromal cells (MSCs) and their differentiation into smooth muscle cells by the supernatant of homogenized bladders in vitro.
Objective: To investigate separation, cultivation and purification of murine marrow mesenchymal stromal cells(MSCs) in vitro and its differentiation into smooth muscle cells by the supernatant of homogenized bladder so as to contribute to the seed cells of bladder tissue engineering.
Methods: MSCs were collected from degermed femurs, tibias and sternum of 4 to 6-week-old SD rat by flushing the shaft with buffer (D-Hank’s, PH 7.2) using a syringe with a No. 26 G needle. Cells were cultivated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 100u/mL penicillin-streptomycin). When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The morphologic characteristics of MSCs were studied by HE staining and scanning electron microscope (SEM). The growth curve was tested by MTT assay. The 4th MSCs were cultured in DMEM with 1% DMSO 8 hours before changing the supernatant of homogenized bladders to induce differentiation. Morphology of cells was observed and immunocytochemistry was performed to detect the expression of specificα-SMA.
Results: The components of primarily cultured MSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clones began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. The HE staining result showed that the cell body was fusiform, and some of them had processes. The big and deep staining basophilia nucleus were in the middle of the cell body. Under SEM we could find three kinds of cell morphologies: the most were the large flattened cells and there were a few spindle-shaped and globe cells. On the cells surface we could see a large amount of short and thick microvillus. The growth curve of P2, P4, P6 were quite similar and the cells biological characters kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase. After induction, MSCs demonstrated smooth muscle cell-like morphology and a hill-and-valley growth pattern as well as expressed the specificα-SMA. Its differentiation rate was (45.6±3.5)% vs control group’s(3.8±0.77)% ,there was significant deviation between the two groups.
Conclusion: 1.In this part, a simple new method which was used for the separation, purification and cultivation in vitro of MSC from SD rat bone marrow by total marrow culture associating with adhering to wall has been established. The MSCs could proliferate immediately and keep their biological character stable in vitro.
2. The cells were noted to have a large expansive potential and a fusiform morphology. The growth curves of P2, P4, P6 were quite similar and the cell biological character kept stable.
3. MSCs could be induced into smooth muscle cells by the supernatant of homogenized bladders in vitro, thus providing sufficient amount of seeds cells to bladder tissue engineering.
Part II: Research the characteristic and preparation of human acellular amniotic membrane graft for tissue engineering applications
Objective:To investigate the preparation method of human acellular amniotic membrane(HAAM) and evaluate the feasibility of using HAAM as biomaterial scaffold to construct tissue engineering bladder.
Methods:Human amniotic membranes were decellularized by the method of enzyme digestion and eradicator washing. The human acellular amniotic membranes (HAAM) were then examined by HE staing and electron microscope to confirm no cell elements remained. Murine marrow mesenchymal stromal cells were implanted in 96-hole-plank and cultured by HAAM extract liquid or normal culture medium. The cytotoxcity of HAAM was tested through MTT assay.6-8 pieces of HAAM were overlapped with fiber trend and exposured under 200W light for 3 hours to increase its thickness and tension.
Results:The HAAM was white and translucent, and the thickness was about 0.1mm. It was lubricous and had good elasticity and toughness. There were no cell elements remained under the examination of optical microscope and electron microscope. Many hole structure and remain figure were seen and rich collagen were observed by scanning electron microscope in HAAM. The cytotoxcity score was I, which indicated that HAAM had a good biocompatibility. The thickness of cross-link HAAM was about 0.6-0.8 mm and its tension was increased.
Conclusion:HAAM can be obtained by enzyme digestion and eradicator washing method. This scaffold has good elasticity and toughness, on which the seeds can adhibit and metabolize. The HAAM has good biocompatibility. The cross-link HAAM is thick and tough to endure the suturation and drag. HAAM can be used as ideal biomaterial scaffold of bladder tissue engineering.
Part III: Construction the tissue engineering bladder and replacement experiment for rat
Objective: To investigate the feasibility of bladder repair and replacement by tissue engineering bladder through the construction of murine tissue engineering bladder by marrow mesenchymal stromal cells(MSCs) and human acellular amniotic membrane graft(HAAMG).
Methods: The MSCs of the rats were implanted in HAAM and cultured for about 5 days. Then the tissue engineering bladders were harvested. The cells were observed with phase contrast microscope and the tissue engineering bladders were observed by HE staining and scanning electron microscope.30 rats were divided into 3 groups randomly and marked with A, B and C. There were 12 animals in group A and B, but 6 animals in group C. Hemicystectomies were performed in all 3 groups. The 12 defects of group A were repaired with the tissue engineering bladders, group B with HAAM grafts and the last 6 defects of group C were sutured directly. The rats underwent postoperative assessment of bladder volume and cystography after 2,4,8 weeks. The animal bladders were obtained after that, through which the tissue regeneration was examined.
Results: There was no evident cell proliferation on the first and second day of coculture of HAAM and MSCs. The cells began to stick to HAAM on the third day and the number of adherent cells increased with the time. On the fifth day of coculture, by HE stain, it could be seen that MSCs adhered to amnion. The cells bodies were bigger, fusiform and uniform. Under scanning electron microscope, there were scattered cells adhered to the surface of material. At 2,4,8 weeks after surgery, the bladder volumes of the group A and B were different significantly as compared with that of the group C(p<0.01), but there is no significant difference between the group A and B(p>0.05 ).The Cystography indicated the morphology of the bladder was normal in A and B group at 8 weeks, but became small in C group. The tissue of the anastomoses area grew well after 2 week in A and B group. Portion of HAAM was absorbed after 2 weeks. The tissue structure of the replacement was somewhat the same as that of the normal one after 4,8 weeks. Through the pathological examination, there were lots of inflammatory cells in the replacement tissue in A and B group after 2 weeks, and these inflammatory cells vanished 8 weeks later. The musculature could be observed in A group after 2 weeks and increased along with the time, but little in B group. The epithelial cells could be observed in both A and B group after 2 weeks and increased with the time.
Conclusion: After transplantation with MSCs/HAAM, epithelial cells and smooth muscle cells can regenerated in the graft, and the graft can be absorbed quickly. The tissue engineering bladder constructed by MSCs and HAAM can be used as an ideal biomaterial to replace and repair the bladder。
引文
1 Pittenger MF, M ackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284(5411):143-7
2 Caplan AI. The mesengenic process. Clin Plast Surg,1994,21(3):429-35
3 Majumdar MD, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs)and stromal cells,J Cell Physiol,1998,176(1):57-66
4 Dennis JE, Merriam A, Awadallah A, et al. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res,1999,14(5):700-709
5 Makino S, Fukuda K, Miyoshi S, et al . Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest ,1999,103 (5) :697-705
6 Wang JS, Shum2Tim D, Chedrawy E, et al . The coronary delivery of marrow stromal cells for myocardial regeneration :pathophysiologic and therapeutic implications. J Thorac Cardiovasc Surg ,2001,122(4) :699-705
7 Jackson KA,Majka SM,Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest,2001,107(11):1395-402
8 Robert E, Schwartz, Morayma Reyes, et al. Verfaillie Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.J.Clin.Invest,2002,109(10):1291-302
9 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res,2000,61(4):364-7
10 Woodbury D, Reynold K, Black IB, et al. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal gene prior to neurogenesis. J Neurosci Res,2002,69(6):908-17
11鄂征主编.组织与细胞培养技术.第2版,北京:北京人民出版社
12 Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science, 2000,287(5457):1442-6
13 Scheffler B, Horn M, Blumcke I, et al. Marrow-mindedness: a perspective on neuropoiesis. Trends Neurosci, 1999, 22(8): 348-357
14 Mckay R. Stem cells in the central nervous system. Science,1997,276 (5309): 66-71
15 Gordon MY, Blackett NM. Reconstruction of the hematopoietic system after stem cell transplantation. Cell Transplant, 1998,7(4):339-344.
16 VanderKooy D, Weiss S. Why stem cells? Science, 2000,287 (5457): 1439-41
17 Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood,2005,105(4): 1815-22
18 Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graftversus-host disease with third party haploidentical mesenchymal stem cells. Lancet,2004,363(9419):1439-41
19 Schoeberlein A, Holzgreve W, Dudler L, et al. Tissue-specific engraftment after in utero transplantation of allogeneic mesenchymal stem cells into sheep fetuses. Am J Obstet Gynecol,2005,192(4):1044-52
20 Chapel A, Bertho JM, Bensidhoum M, et al. Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome. J Gene Med,2003,5(12):1028-38
21 Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood,2005,106(5):1755-61
22 Haynesworth SE, Goshima J, Goldberg VM, et al. Characterization of cells with osteogenic potential from human marrow. Bone,1992,13(1):81-8
23 Lu L, Zhao C, Liu Y, et al. Therapeutic benefit of TH-engineering mesenchymal stem cells for Parkinson’s disease. Brain Res Brain Res Protoc,2005,15(1):46-51
24 Conget PA ,Minguell JJ. Pheno typical and functional properties of human bone marrow mesenchymal progenitor cells . J Cell Physiol,1999,181 (1) : 67-73
25 Wakitani S, Saito T, Caplan AT. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve,1995,18(12):1417-22
26 Fukuda F. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artifograns,2001,25(3):187-93
27 Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. Clin Invest,1999,103(5):697-705
28 Alexandrina B, Ana-Maria R, Horia M,et al. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells.European Journal of Cell Biology,2008,78(3):173-84
29陈晓东,董海,周之德.骨髓间充质干细胞体外转化为骨骼肌细胞的初步观察.中华手外科杂志,2004,20(4):230-2
30杨景全,路来今,闫俊,等.大鼠脊髓匀浆上清液对骨髓间充质干细胞的诱导分化作用.中国老年学杂志,2007;27(2):245-6
31张金明,何涛,黄红军.骨髓间充质干细胞体外诱导分化为平滑肌细胞的实验.中国临床康复,2006,10(1):23-6
32苗莉,何国祥,景涛,等.观察大鼠血管平滑肌细胞与间充质干细胞直接接触培养对细胞的诱导分化.第三军医大学学报,2007,29(9):814-6
33徐正云,马爱群,王亭忠,等.细胞间接触诱导大鼠骨髓间充质干细胞分化为平滑肌细胞.中国心血管杂志,2005,10(6):416-9
34韩雅玲,康建,李少华.成年鼠骨髓间充质细胞在体外培养中分化为平滑肌细胞.中华医学杂志,2003,83(9):778-81
35 Song YS, Lee HJ, Park IH, et al. Potential differentiation of humanmesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells. Int J Impot Res,2007,19(4):378-85
36 Tomita S, Li RK, Weisel Rd, et al. Autologous transplantation of bone marrow cells improve damaged heart function.Circulation,1999,100(Suppl 19):247-50
37张覃沐.抗肿瘤药物的药理与临床应用.郑州:河南医科大学出版社,1999,361
38 Rephael i A, Aviram A, Rabizadeh E, et al. The role of calcium in differentiation of leukemic cell lines. Cancer Biochem Biophys,1990,11(2):119-25
39尹青,陈炜,赵昱,等.二甲基亚砜对P19细胞体外分化为心肌细胞的影响.细胞生物学杂志, 2007,29:579-84
1 GB/T 16886.12—2000,医疗器械生物学评价的第十二部分:样品制备与参照样品.医疗器械生物学评价标准汇编.中国标准出版社, 2004,80-88
2 GB/T 16886.5—2003,医疗器械生物学评价,第五部分:体外细胞毒性试验.医疗器械生物学评价标准汇编.中国标准出版社, 2004, 186-197
3 Current methods in biomaterial testing and risk assessment Clinical hemorheology Klein CL, Holger K, Mike O Volume: 16, Issue: 2, March 4, 1996, pp. 210
4 Langer R, V acanti JP. Tissue engineering. Science,1993,260(5110):920-926
5 Beiko DT, Knudsen BE, Watterson JD, et al. Urinary tract biomaterials. J Urol,2004,171(6 Pt 1): 2438–44
6 Stephen F, Badylak. The extracellular matrix as a scaffold for tissue reconstruction. Cell & development biology,2002,13(5): 377–83
7 Badylak SF, Lantz GC. Small intestinal submucosa as a large diameter vascular graft in the dog. J Surg Res,1989,47(1): 74-80
8 Cook AD, Hrkach JS, Gao NN, et al. Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial. J Biomed Mater Res,1997,35(4):513-523
9 Barrera DA, Zylstra E, Lansbury PT, et al. Co-polymerization and degradation of poly(lactic acid-co-lysine). Macromolecules,1995, 28:425-432
10 Oberpenning F, Meng J, Yoo JJ, et al. De novo reconstruction of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol,1999,17:149-155
11 Grillo HC, Mckhann CF. The acceptance and evolution of dermal homografts freed of viable cells. Transplantation,1964,2:48-59
12 Gilberta TW, Sellaroa TL, Badylak SF. Thomas W, Gilberta,b, Tiffany L. Decellularization of tissues and organs. Biomaterials,2006,27(19):3675-83
13 Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J Struct Biol,2003,143(3):201-208
14 Dahl SL, Koh J, Prabhakar V, et al. Decellularized native and engineered arterial scaffolds for transplantation. Cell Transplant,2003,12(6):659-66
15 Freytes DO, Badylak SF, Webster TJ, et al. Biaxial strength of multilaminated extracellular matrix scaffolds. Biomaterials,2004,25(12):2353-61
16 Lin P, Chan WC, Badylak SF, Bhatia SN. Assessing porcine liverderived biomatrix for hepatic tissue engineering. Tissue Eng,2004,10(7-8):1046-53
17 Jackson DW, Grood ES, Arnoczky SP, et al. Cruciate reconstruction using freeze dried anterior cruciate ligament allograft and a ligament augmentation device (LAD). An experimental study in a goat model. Am J Sports Med,1987,15(6):528-38
18 Jackson DW, Grood ES, Arnoczky SP, et al. Freeze dried anterior cruciate ligament allografts. Preliminary studies in a goat model. Am J Sports Med,1987,15(4):295-303
19 Woods T, Gratzer PF. Effectiveness of three extraction techniques in the development of a decellularized bone–anterior cruciate ligament-bone graft. Biomaterials,2005,26(35):7339-49
20 Vyavahare N, Hirsch D, Lerner E, et al. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms. Circulation,1997,95(2):479-88
21 Bader A, Schilling T, Teebken OE, et al. Tissue engineering of heart valves-human endothelial cell seeding of detergent acellularized porcine valves. Eur J Cardiothorac Surg,1998,14(3):279-84
22 Gamba PG, Conconi MT, Lo Piccolo R, et al. Experimental abdominal wall defect repaired with acellular matrix. Pediatr Surg Int, 2002,18(5-6):327-31
23 Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible“off the shelf”biomaterial for urethral repair. Urology,1999,54(3):407-10
24 Rieder E, Kasimir MT, Silberhumer G, et al. Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. J Thorac Cardiovasc Surg,2004,127(2):399-405
25 Martins-Green M, Bissel MF. Cell-extracellular matrix interactions in development. Semin Dev Biol,1995,6:149-159
26 Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J Struct Biol, 2003,143(3):201-8
27 Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta,2004,1666(1-2):105-17
28 Barnham JJ, Roper-Hall MJ. Keratoprosthesis: a longterm review. Br J Ophthalmol,1983,67(7):368-76
29文道源,李舒梅,林卡莉.新鲜羊膜、冻干羊膜、羊膜细胞外基质的形态研究.中国实用眼科杂志,2003,23(10):734-8
30 Koizumi N, Inatomi T, Quantock AI, et al. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea,2000,19(1):65-71
31 Cho HJ, Djalilian AR, Obritsch WF, et al. Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentinte into corneal epithelial-type cells.Cornea,1999,18(2):216-24
32 Liotta LA, Lee CW, Morakis DJ, et al. New method for preparing large surfaces of intact human basement for tumor invasion studies. Cancer Lett,1980,11(2):141-52
33 Goto Y, Noguchi Y, Nomura A, et al. In vitro reconstruction fo the tracvheal epithelium . Am J Respir Cell Mol Biol,1999,20(2):312-8
34陈有刚,朱家恺.人羊膜基底膜桥接神经缺损实验研究.中华显微外科,1990,13(1):20-24
35罗静聪,李秀群,杨志明,等.脱细胞羊膜的制备及其生物相容性研究.中国修复重建外科杂志,2004,18(2):108-11
36 Davis CE, Blaker SN, Engvall E, et al. Human amnion membrane serve as substratum for growing axon in vivo and vitro. Science,1987,236(4805):1106-9
37王能兴,朱争艳,姚爱琳,等.以羊膜为载体培养游离软骨细胞修复兔关节软骨缺损.生物医学工程与临床.2002,6(4):185-8
38 Cui Q, Harvery AR. At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats. J Neurosci,1995,15(12):8143-55
39 Ban Y, Cooper LJ, Fullwood NJ, et al. Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting. Exp-Eye Res,2003,76(6):735-43
40 Meller D, Dabul V, Tseng SC. Expansion of conjunctival epithelial progenitor cells on amniotic membrane. Exp Eye Res,2002,74(4):537-45
41 Ohno-M, atsui K, Ichinose S,et al. The effects of amniotic membrane on retinal pigment epithelial cell differentiation. Mol Vis,2005,11:1-10
42 Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology,2001,108(9):1569-74
43闫国和,栗永萍,艾国平,等.人羊膜负载猪角脘细胞重建表皮的形态学研究.第三军医大学学报,2002,24(8):933-6
44闫国和,栗永萍,艾国平,等.人羊膜负载猪角骨髓间充质干细胞生长的形态学研究.第三军医大学学报,2002,24(7):775-7
45 Kallenbach K, Fernandez IIA, Seghezzi G, et al. A quantitative in vitro model of smooth muscle cell migration through the arterial wall using the human amniotic membrane. A rterioscler Thromb Vasc Biol,2003,23(6):1008-13
46 Tryphonopoulos P, Espana EM, Tseng SC, et al. Rat enterocyte cultures on human amniotic membranes. Transplant Proc,2004,36(2):373-4
47 Zhao LP, Chan RMW. Quantitative analysis of neural growth on various substrata especially the human amniotic basement membrane by Eliserrender. Acta Anatomica Sinica,1989,20:71-6
48 Grauss RW, Hazekamp MG, Oppenhuizen F, et al. Histological evaluation of decellularised porcine aortic valves: matrix changes due to different decellularisation methods. Eur J Cardiothorac Surg, 2005,27(4):566-71
49 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983,65(1-2):55-63
1 Yoo JJ, Oberpenning F, Atala A. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology,1998,51(2):221-5
2 Chung SY, Krivorov NP, Rausei V, et al. Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. J Urol 2005, 174(1):353-9
3 Zhang Y, Lin HK, Frimberger D, et al. Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. BJU Int 2005,96(7):1120-5
4 Oberpenning F, Meng J, Yoo JJ and Atala A: De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999,17(2):149-55
5 Atala A, Bauer SB, Soker S, et al. Tissueengineered autologous bladders for patients needing cystoplasty. Lancet 2006,367(9518): 1241-6
6 Zhang Y, Frimberger D, Cheng EY, et al. Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model. BJU Int 2006,98(5):1100-5
7 Badylak SF, Kropp B, McPherson T, et al. Small intestinal submucosa: a rapidly resorbed bioscaffold for augmentation cystoplasty in a dog model. Tissue Eng 1998,4(4):379-87
8 Kropp BP, Rippy MK, Badylak SF, et al. Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J Urol 1996,155(6):2098-104
9 Folkman J, Hochberg MM. Self regulation of growth in three dimensions. J Exp Med,1973,138(4):745-53
10 Atala A, Vacanti JP, Peters CA, et al. Formation of urothelial structures invivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. J Urol,1992,148(2 Pt 2):658-62
11 AtalaA. Bladder regeneration by tissue engineering. BJUInt,2001,88(7):765-70
12 YooJJ,AtalaA. Tissue engineering of genitourinary organs. Ernst Schering Res Found Workshop,2002,(35):105-27
13 Brown AL, Tamara T, Brook-Allred, et al. Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle–urothelial cell interactions. Biomaterials,2005,26(5):529-43
14 Kim BS, Putnam AJ, Kulik TJ. Optimizing seeding and culture methods to engineering smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng,1998,57(1):46-54
15 Qiu QQ,Ducheyne P,Ayyaswamy PS. 3D bone tissue engineered with bioactive microsphers in simulated microgravity. In Vitro Cell Dev Biol Anim,2001,37(3):157-65
16 Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials,2006,27(19):3675-83
17 Liang HC, Chang Y, Hsu CK, et al. Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern. Biomaterials,2004,25(17):3541-52
18 Munting E, Wilmart JF, Wijne A, et al. Effect of sterilization on osteoinduction. Comparison of five methods in demineralized rat bone. Acta Orthop Scand,1988,59(1):34-8
19 Thomas W, Gilbert, Ann M, et al. A quantitative method for evaluating the degradation of biologic scaffold materials. Biomaterials,2007,28(2): 147-150
20 Record RD, Hillegonds D, Simmons C, et al. In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair. Biomaterials,2001,22(19):2653-9
21 Gilbert TW, Stewart-Akers AM, Simmons-Byrd A, et al. Degradation and remodeling of small intestinal submucosa in Achilles tendon repair. J BoneJoint Surg (US),2007,89(3),621-30
22 Rebel JM, de Boer WI, Thijssen CD, et al. An in vitro model of intra-epithelial expansion of transformed urothelial cells. Int J Cancer,1993,54(5):846-50
23 Sutherland RS, Baskin LS, Hayward SW, et al. Regeneration of bladder urothelium, smooth muscle, blood vessels and nerves into an acellular tissue matrix. J Urol,1996,156(2 Pt 2):571-7
24 Mcdevitt CA, Wildey GM, Cutrone RM. Transforming growth factor- beta1 in a sterilized tissue derived from the pig small intestine submucosa. J Biomed Mater Res A,2003,67(2):637-40
25 Hurst RE, Bonner RB. Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence microscopy. J Biomater Sci Polym Ed,2001,12(11):1267-79
26 Lu SH, Sacks MS, Chung SY, et al. Biaxial mechanical properties of muscle-derived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials,2005,26(4):443-9
27 Perchota HJ , Gleason CA ,Dahms SE, et al. Bladder acellular matrix graft: in vivo functional properties of the regenerated rat bladder. Urol Res,1999,27(3):206-13
28 Nuininga JE, van Moerkerk H, Hanssen A, et al. A rabbit model to tissue engineer the bladder. Biomaterials,2004,25(9):1657-61
1 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-7
2 Wakitani S, Saito T, Caplan AT. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.Muscle Nerve,1995,18(12):1417-22
3 Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. Clin Invest,1999,103(5):697-705
4 Fukuda F. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artifograns,2001,25(3):187-93
5孙丽莉,谭玉珍,王海杰,等.骨髓间充质干细胞的克隆培养及其向心肌细胞的诱导分化.复旦学报(医学版),2003,30(6):519-22
6 Liu Y, Song J, Liu W,et al. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyo-genic differentiation. Cardiovasc Res,2003,58(2):460-8
7 Ye NS, Chen J, Luo GA, et al. Proteomic profiling of rat bone marrow mesenchymal stem cells induced by 5-azacytidine. Stem Cells Dev,2006,159(4):665-76
8 Ye NS, Zhang RL, Zhao YF, et al. Effect of 5-azacytidine on the protein expression of porcine bone marrow mesenchymal stem cells in vitro[J]. Genom Proteom Bioinform,2006,4(1):18-25
9 Tomita S, Mickle DA, Weisel RD,et al. Improved heart function with myogenesis and Angiogenesis after autologous porcine bone marrow stromal cell transplantation. Thorac Cardiovasc Surg,2002,123(6):1132-40
10 Tomita S,Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improve damaged heart function.Circulation,1999,100[Supp19]:247-56
11 Xu W, Zhang X, Qian H, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. ExpBiol Med(Maywood),2004,229(7):623-31
12 Rangappa S, Entwistle JW, Wechsler AS, et al. Cardiomyocyte mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. Thorac Cardiovasc Surg,2003,126(1):124-32
13 Shim WS, Jiang S, Wong P, et al. Exvivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells. Biochem BiophysResCommun,2004,324(2):481-8
14 Li H, Yu B, Zhang Y, et al. Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem BiophysResCommun,2006,341(2):320-5
15 Shake JG, Gruber JP, Baumgartner WA, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model:Engraftment and functional affects.Ann Thorae Surg 2002,73(6):1919-26
16 Bayes-Genis A, Roura S, Soler-Botija C, et al. Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cell. Transplant Proc,2005,37(8):4077-9
17 Zhang FB,LI L,Fang B,et al. Passage-restricted differentiation Potential of mesenchymal stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun,2005,336(3):784-92
18 Liu Y, Song J, Liu W, et al. Growth and differentiation of rat bone marrow stromal cells:Does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003,58:460-8
19 Ninomiya K, Takahashi A, Fujioka Y, et al. Transforming growth factor-beta signaling enhances transdifferentiation of macrophages into smooth muscle-like cells. Hyertens Res,2006,29:269-76
20 Alexandrina B, Ana-Maria R, Horia M,et al. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells. European Journal of Cell Biology,2008,78(3):173-84
21 Narita Y, Yamawaki A, Kagami H, et al. Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle celllineage.Cell Tissue Res,2008,333(3):449-59
22 Yoon BS, Yoo SJ, Lee JE, et al. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. Differentiation,2006,74:149-59
23 Wu YC, Cui L, Li G, et al. PDGF-BB initiates vascular smooth muscle-like phenotype differentiation of human bone marrow mesenchymal stem cells in vitro. Zhonghua Zheng Xing Wai Ke Za Zhi,2007,23(4):335-9
24孟寒,阮秋蓉,瞿智玲,等.小鼠骨髓间充质干细胞体外诱导向平滑肌样细胞分化过程中myocardin的表达.中国动脉硬化杂志,2008,16(1):13-16
25张金明,何涛,黄红军.骨髓间充质干细胞体外诱导分化为平滑肌细胞的实验.中国临床康复,2006,10(1):23-26
26 Brown LF, Detmar M, Tognazzi K, et al. Uterine smooth cells express functional receptors (flt-1 and KDR)for vascular permeability factor vascular endothelial growth factor. Lab Invest,1997,76(2):245-55
27 Liu X,Lin CS, Graziottin T,et al. Vascular endothelial growth factor promotes proliferation and migration of cavernous smooth muscle cells.Urol,2001,166(1):354-60
28 Pueyo ME, Chen Y, DAngelo G, et al. Regulation of vascular endothelial growth factor expression by cAMP in rat aortic smooth muscle cells. Exp Cell Res,1998,238(2):354-8
29苗莉,何国祥,景涛等.观察大鼠血管平滑肌细胞与间充质干细胞直接接触培养对细胞的诱导分化.第三军医大学学报,2007,29(9):814-6
30徐正云,马爱群,王亭忠,等.细胞间接触诱导大鼠骨髓间充质干细胞分化为平滑肌细胞.中国心血管杂志,2005,(10)6:416-9
31韩雅玲,康建,李少华.成年鼠骨髓间充质细胞在体外培养中分化为平滑肌细胞.中华医学杂志,2003,(83)9:778-81
32 Song YS, Lee HJ, Park IH, et al. Potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells. Int J Impot Res,2007,19(4):378-85
33 Kobayashia N,Yasu T,Ueba H,et al. Mechanical stress promotes theexpression of smooth muscle-like properties in marrow stromal cells. Exp Hematol,2004,32(12):1238-45
34 Park JS,Chu JSF,Cheng C,et al. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnol Bioemg,2004,88(3):359-67
35 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-7
36陈晓东,董海,周之德.骨髓间充质干细胞体外转化为骨骼肌细胞的初步观察.中华手外科杂志,2004,20(4):230-2
37 Lattanzi L, Salvatori G, Coletta M, et al. High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. Clin invest,1998,101(10):2119-28
38 Edmondson DG, Olson EN. Helix-loop-helix proteins as regulators of muscle-specific transcription. Biol Chem.,1993,268(22):755-8
39张勇,邹仲敏,郭朝华,等. MyoD基因诱导骨髓间充质干细胞分化为成肌细胞的实验研究.第三军医大学学报,2002,24(12):1423-6
40孙占胜,陈振强.成肌分化因子和5-氮杂胞苷联合诱导骨髓间充质干细胞向骨骼肌细胞分化的实验研究.中国修复重建外科杂志,2007,21(12):1371-5
41曾缨,王凌云.骨髓间质干细胞诱导为肌样细胞分化相关基因的表达.中国病理生理杂志,2006,22(9):1665-8
42 Sun Z, Chen Z, Wang B. In vitro differentiation of rat mesenchymal stem cells into skeletal muscle cells induced by myoblast differentiation factor and 5-azacytidine.Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi,2007,21(12):1371-5
43 Rogers JJ,Young HE,Adkison LR,et al. Differentiation factors induce expression of muscle、fat、cartilage and bone in a clone of mouse pluripotent mesenchymal stem cells. Am Surg,1995,61(3):231-6
44 Stocum DL. Development. A trail of transdifferentiation. Science,2002,298(5600):1901-3
45 Chan J,O’Donoqhue K, Gavina M, et al. Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration.Stem Cells,2006,24(8):1879-91
1 Stock U A, Vacanti J P. Tissue engineering: current state and prospects. Annu RevMed,2001,52:443-51
2 Allman A J, Mcpherson T B, Badylak S F, et al. Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response.Transplantation,2001,71(11):1631-40
3 Badylak S, Kokini K, Tullius B,et al. Morphologic study of small intestinal sub mucosa as a body wall repair device. Surg Res,2002,103(2):190-202
4 Kropp BP, Eppley BL, Prevel CD, et al. Experimental assessment of small intestinal submucosa as a bladder wall substitute. Urol,1995,46(3):396-400
5 Kropp BP, Rippy MK, Badylak SF, et al. Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. Urol,1996,155(6):2098-104
6 Record RD, Hillegonds D, Simmons C, et al. In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair. Biomaterials,2001,22(19):2653-9
7 Nuininga J E, Moerkerk H v, Hanssen A, et al. A rabbit model to tissue engineer the bladder. Biomaterials,2004,25(9):1657-61
8 Schultheiss D, Gabouev A I, Cebotari S, et al. Biological vascularizedmatrix for bladder tissue engineering. matrix preparation, reseeding technique and short-term implantation in a porcine model. Urol,2005,173(1):276-80
9 Campodonico R, Benelli A, Michelazzi E, et al. Bladder cell culture on small intestinal submucosa as bioscaffold:experimental study on engineered urothelial grafts. Eur Urol,2004,46(4):531-7
10 Dhams SE, Piechota HJ, Dahiya R, et al. Composition and biome chemical properties of the bladder acellular matrix graft: comparative analysis in rat、pig and human. Br J Urol,1998,82(3):411-9
11 Probst M, Dahiya R, Carrier S, et al. Reproduction of functional smooth muscle tissue and partial bladder replacement. Br J Urol,1997,79(4):505-15
12 Atala A. Technology insight: Applications of tissue engineering and biological substitutes in urology. Nat Clin Pract Urol, 2005,2(3):143-9
13 Wefer J, Sievert K D, Schlote N, et al. Time dependent smooth muscle regeneration and maturation in a bladder acellular matrix graft: Histological studies and in vivo functional evaluation. Urol,2001,165(5):1755-9
14 Reddy P P, Barrieras D J, Wilson G, et al. Regeneration of functional bladder substitutes using large segment acellular matrix allografts in a porcinemodel.Urol,2000,164(3Pt2):936-41
15 Yoo JJ, Meng J, Oberpenning F, et al. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology,1998,51(2):221-5
16 Brown A L, Fathat W, Merguerian P A, et al. 22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model.Biomaterials,2002,23(10):2179-90
17韩平,宋超,魏强,等.组织工程膀胱细胞外基质生物相容性的实验研究.四川大学学报,2007,38(6):1009-12
18 Hattori K, Joraku A, Miyagawa T ,et al. Bladder reconstruction using a collagen patch prefabricated within the omentum. Int J Urol,2006,13(5):529-37.
19杨中华,王行环,王怀鹏,等.以纤维蛋白凝胶为支架构建组织工程胃新膀胱的研究.现代泌尿外科杂志,2007, 12(5):305-7
20 Atala A, Bauer S B, Soker S, et al. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet,2006,367:1241-6
21 Chung S Y. Bladder tissue-engineering: a new practical solution? Lancet,2006,367:1215-6
22 Shinoka T, Shum-Tim D, Ma PX, et al. Creation of viable pulmonary artery autografts through tissue engineering. Thorac Cardiovasc Surg,1998,115(3):536-46
23 Scheepens WA, Debie RA, Weil EH, et al. Unilateral versus bilateral sacral neuromodulation in patients with chronic voiding dysfunction.Urol,2002,168(5):2046-50
24 Cook AD, Hrkach JS, Gao NN, et al. Characterization and development of RGD-peptide-modied? poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial. J Biomed Mater Res, 1997,35:513-23
25 Barrera DA, Zylstra E, Lansbury PT, et al. Co-polymerization and degradation of poly(lactic acid-co-lysine). Macromolecules,1995, 28:425-32
26 Oberpenning F, Meng J, Yoo JJ, et al. De novo reconstruction of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol,1999,17:149-55
27 Atala A. Tissue engineering for bladder substitution. World J Urol,2000,18(5):364-70
28 Kirk D, Kagawa S, Vener G, et al. Selective growth of normal adult human urothelial cells in serum-free medium. In Vitro Cell Dev Biol 1985,21(3pt1):165-71
29 Cilento BG, Freeman MR, Schneck FX, et al. Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro.Urol,1994,152:655-70
30杨中华,王行环,杨梅超,等.猪膀胱移行上皮细胞培养及其在纤维蛋白凝胶表面生长的评价.中国临床康复,2006,10(17),61-4
31 Baskin LS , Howard PS, Duckett JW, et al. Bladder smooth muscle cells in culture: identification and charactarization. J Urol,1993,149:190-7
32 Sui GP, Wu C, Fry CH. The electrophysiological properties of cultured and freshly isolated detrusor smooth muscle cells. J Urol,2001, 165:627-33
33卢慕峻,王忠,傅强,等.组织工程化膀胱平滑肌结构的体外构建.组织工程与重建外科杂志,2006,2(2),76-8
34 Chung SY, Krivorov NP, Rausei V, et al. Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. Urol,2005,174(1):353-9
35 Schultheiss D, Gabouev A, Pilatz A, et al. Use of bone marrow derived mesenchymal stem cells for autologous tissue engineering of urinary bladder muscularis. European Urology Supplements, 2004, 3:159
36 Frimberger D, Morales N, Gearhart J, et al. Human embryoid body-derived stem cells in tissue engineering—enhanced migration in co-culture with bladder smooth muscle and urothelium.Urol,2006,67(6),1298-303
37 Lakshmanan Y ,Frimberger D ,Gearhart J, et al. Human embryoid body-derived stem cells in co-culture with bladder smooth muscle and urothelium.Urol,2005,65(4):821–6
38姚康德,宋雪峰,刘文广,等.组织工程研究与开发进展.中国修复重建外科杂志,2002,16(5):325-8
39 Kim BS, Putnam AJ, Kulik TJ. Optimizing seeding and culture methods to engineering smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng,1998,57(1):46-54
40 Qiu QQ,Ducheyne P,Ayyaswamy PS. 3D bone tissue engineered with bioactive microsphers in simulated microgravity. In Vitro Cell Dev Biol Anim,2001,37(3):157-65
41 Fauza Do, Fishman S, Mehegan K, et al. Videofeto-scopically assisted fetal tissue engineering: Bladder augmentation. Ped Surg,1998,33:7-12
42 Zhang Y, Kropp BP,Moore P,et al. Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa:potential applications for tissue engineering technology.Urol,2000,164(3Pt2):928-5
43 Danielsson C, Ruault S, Basset-Dardare A ,et al. Modified collagen fleece,a scaffold for transplantation of human bladder smooth muscle cells.Biomaterials,2006,27(7):1054-60
2 Caplan AI. The mesengenic process. Clin Plast Surg,1994,21(3):429-35
3 Majumdar MD, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs)and stromal cells,J Cell Physiol,1998,176(1):57-66
4 Dennis JE, Merriam A, Awadallah A, et al. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res,1999,14(5):700-709
5 Makino S, Fukuda K, Miyoshi S, et al . Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest ,1999,103 (5) :697-705
6 Wang JS, Shum2Tim D, Chedrawy E, et al . The coronary delivery of marrow stromal cells for myocardial regeneration :pathophysiologic and therapeutic implications. J Thorac Cardiovasc Surg ,2001,122(4) :699-705
7 Jackson KA,Majka SM,Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest,2001,107(11):1395-402
8 Robert E, Schwartz, Morayma Reyes, et al. Verfaillie Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.J.Clin.Invest,2002,109(10):1291-302
9 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res,2000,61(4):364-7
10 Woodbury D, Reynold K, Black IB, et al. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal gene prior to neurogenesis. J Neurosci Res,2002,69(6):908-17
11鄂征主编.组织与细胞培养技术.第2版,北京:北京人民出版社
12 Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science, 2000,287(5457):1442-6
13 Scheffler B, Horn M, Blumcke I, et al. Marrow-mindedness: a perspective on neuropoiesis. Trends Neurosci, 1999, 22(8): 348-357
14 Mckay R. Stem cells in the central nervous system. Science,1997,276 (5309): 66-71
15 Gordon MY, Blackett NM. Reconstruction of the hematopoietic system after stem cell transplantation. Cell Transplant, 1998,7(4):339-344.
16 VanderKooy D, Weiss S. Why stem cells? Science, 2000,287 (5457): 1439-41
17 Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood,2005,105(4): 1815-22
18 Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graftversus-host disease with third party haploidentical mesenchymal stem cells. Lancet,2004,363(9419):1439-41
19 Schoeberlein A, Holzgreve W, Dudler L, et al. Tissue-specific engraftment after in utero transplantation of allogeneic mesenchymal stem cells into sheep fetuses. Am J Obstet Gynecol,2005,192(4):1044-52
20 Chapel A, Bertho JM, Bensidhoum M, et al. Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome. J Gene Med,2003,5(12):1028-38
21 Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood,2005,106(5):1755-61
22 Haynesworth SE, Goshima J, Goldberg VM, et al. Characterization of cells with osteogenic potential from human marrow. Bone,1992,13(1):81-8
23 Lu L, Zhao C, Liu Y, et al. Therapeutic benefit of TH-engineering mesenchymal stem cells for Parkinson’s disease. Brain Res Brain Res Protoc,2005,15(1):46-51
24 Conget PA ,Minguell JJ. Pheno typical and functional properties of human bone marrow mesenchymal progenitor cells . J Cell Physiol,1999,181 (1) : 67-73
25 Wakitani S, Saito T, Caplan AT. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve,1995,18(12):1417-22
26 Fukuda F. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artifograns,2001,25(3):187-93
27 Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. Clin Invest,1999,103(5):697-705
28 Alexandrina B, Ana-Maria R, Horia M,et al. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells.European Journal of Cell Biology,2008,78(3):173-84
29陈晓东,董海,周之德.骨髓间充质干细胞体外转化为骨骼肌细胞的初步观察.中华手外科杂志,2004,20(4):230-2
30杨景全,路来今,闫俊,等.大鼠脊髓匀浆上清液对骨髓间充质干细胞的诱导分化作用.中国老年学杂志,2007;27(2):245-6
31张金明,何涛,黄红军.骨髓间充质干细胞体外诱导分化为平滑肌细胞的实验.中国临床康复,2006,10(1):23-6
32苗莉,何国祥,景涛,等.观察大鼠血管平滑肌细胞与间充质干细胞直接接触培养对细胞的诱导分化.第三军医大学学报,2007,29(9):814-6
33徐正云,马爱群,王亭忠,等.细胞间接触诱导大鼠骨髓间充质干细胞分化为平滑肌细胞.中国心血管杂志,2005,10(6):416-9
34韩雅玲,康建,李少华.成年鼠骨髓间充质细胞在体外培养中分化为平滑肌细胞.中华医学杂志,2003,83(9):778-81
35 Song YS, Lee HJ, Park IH, et al. Potential differentiation of humanmesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells. Int J Impot Res,2007,19(4):378-85
36 Tomita S, Li RK, Weisel Rd, et al. Autologous transplantation of bone marrow cells improve damaged heart function.Circulation,1999,100(Suppl 19):247-50
37张覃沐.抗肿瘤药物的药理与临床应用.郑州:河南医科大学出版社,1999,361
38 Rephael i A, Aviram A, Rabizadeh E, et al. The role of calcium in differentiation of leukemic cell lines. Cancer Biochem Biophys,1990,11(2):119-25
39尹青,陈炜,赵昱,等.二甲基亚砜对P19细胞体外分化为心肌细胞的影响.细胞生物学杂志, 2007,29:579-84
1 GB/T 16886.12—2000,医疗器械生物学评价的第十二部分:样品制备与参照样品.医疗器械生物学评价标准汇编.中国标准出版社, 2004,80-88
2 GB/T 16886.5—2003,医疗器械生物学评价,第五部分:体外细胞毒性试验.医疗器械生物学评价标准汇编.中国标准出版社, 2004, 186-197
3 Current methods in biomaterial testing and risk assessment Clinical hemorheology Klein CL, Holger K, Mike O Volume: 16, Issue: 2, March 4, 1996, pp. 210
4 Langer R, V acanti JP. Tissue engineering. Science,1993,260(5110):920-926
5 Beiko DT, Knudsen BE, Watterson JD, et al. Urinary tract biomaterials. J Urol,2004,171(6 Pt 1): 2438–44
6 Stephen F, Badylak. The extracellular matrix as a scaffold for tissue reconstruction. Cell & development biology,2002,13(5): 377–83
7 Badylak SF, Lantz GC. Small intestinal submucosa as a large diameter vascular graft in the dog. J Surg Res,1989,47(1): 74-80
8 Cook AD, Hrkach JS, Gao NN, et al. Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial. J Biomed Mater Res,1997,35(4):513-523
9 Barrera DA, Zylstra E, Lansbury PT, et al. Co-polymerization and degradation of poly(lactic acid-co-lysine). Macromolecules,1995, 28:425-432
10 Oberpenning F, Meng J, Yoo JJ, et al. De novo reconstruction of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol,1999,17:149-155
11 Grillo HC, Mckhann CF. The acceptance and evolution of dermal homografts freed of viable cells. Transplantation,1964,2:48-59
12 Gilberta TW, Sellaroa TL, Badylak SF. Thomas W, Gilberta,b, Tiffany L. Decellularization of tissues and organs. Biomaterials,2006,27(19):3675-83
13 Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J Struct Biol,2003,143(3):201-208
14 Dahl SL, Koh J, Prabhakar V, et al. Decellularized native and engineered arterial scaffolds for transplantation. Cell Transplant,2003,12(6):659-66
15 Freytes DO, Badylak SF, Webster TJ, et al. Biaxial strength of multilaminated extracellular matrix scaffolds. Biomaterials,2004,25(12):2353-61
16 Lin P, Chan WC, Badylak SF, Bhatia SN. Assessing porcine liverderived biomatrix for hepatic tissue engineering. Tissue Eng,2004,10(7-8):1046-53
17 Jackson DW, Grood ES, Arnoczky SP, et al. Cruciate reconstruction using freeze dried anterior cruciate ligament allograft and a ligament augmentation device (LAD). An experimental study in a goat model. Am J Sports Med,1987,15(6):528-38
18 Jackson DW, Grood ES, Arnoczky SP, et al. Freeze dried anterior cruciate ligament allografts. Preliminary studies in a goat model. Am J Sports Med,1987,15(4):295-303
19 Woods T, Gratzer PF. Effectiveness of three extraction techniques in the development of a decellularized bone–anterior cruciate ligament-bone graft. Biomaterials,2005,26(35):7339-49
20 Vyavahare N, Hirsch D, Lerner E, et al. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms. Circulation,1997,95(2):479-88
21 Bader A, Schilling T, Teebken OE, et al. Tissue engineering of heart valves-human endothelial cell seeding of detergent acellularized porcine valves. Eur J Cardiothorac Surg,1998,14(3):279-84
22 Gamba PG, Conconi MT, Lo Piccolo R, et al. Experimental abdominal wall defect repaired with acellular matrix. Pediatr Surg Int, 2002,18(5-6):327-31
23 Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible“off the shelf”biomaterial for urethral repair. Urology,1999,54(3):407-10
24 Rieder E, Kasimir MT, Silberhumer G, et al. Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. J Thorac Cardiovasc Surg,2004,127(2):399-405
25 Martins-Green M, Bissel MF. Cell-extracellular matrix interactions in development. Semin Dev Biol,1995,6:149-159
26 Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J Struct Biol, 2003,143(3):201-8
27 Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta,2004,1666(1-2):105-17
28 Barnham JJ, Roper-Hall MJ. Keratoprosthesis: a longterm review. Br J Ophthalmol,1983,67(7):368-76
29文道源,李舒梅,林卡莉.新鲜羊膜、冻干羊膜、羊膜细胞外基质的形态研究.中国实用眼科杂志,2003,23(10):734-8
30 Koizumi N, Inatomi T, Quantock AI, et al. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea,2000,19(1):65-71
31 Cho HJ, Djalilian AR, Obritsch WF, et al. Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentinte into corneal epithelial-type cells.Cornea,1999,18(2):216-24
32 Liotta LA, Lee CW, Morakis DJ, et al. New method for preparing large surfaces of intact human basement for tumor invasion studies. Cancer Lett,1980,11(2):141-52
33 Goto Y, Noguchi Y, Nomura A, et al. In vitro reconstruction fo the tracvheal epithelium . Am J Respir Cell Mol Biol,1999,20(2):312-8
34陈有刚,朱家恺.人羊膜基底膜桥接神经缺损实验研究.中华显微外科,1990,13(1):20-24
35罗静聪,李秀群,杨志明,等.脱细胞羊膜的制备及其生物相容性研究.中国修复重建外科杂志,2004,18(2):108-11
36 Davis CE, Blaker SN, Engvall E, et al. Human amnion membrane serve as substratum for growing axon in vivo and vitro. Science,1987,236(4805):1106-9
37王能兴,朱争艳,姚爱琳,等.以羊膜为载体培养游离软骨细胞修复兔关节软骨缺损.生物医学工程与临床.2002,6(4):185-8
38 Cui Q, Harvery AR. At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats. J Neurosci,1995,15(12):8143-55
39 Ban Y, Cooper LJ, Fullwood NJ, et al. Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting. Exp-Eye Res,2003,76(6):735-43
40 Meller D, Dabul V, Tseng SC. Expansion of conjunctival epithelial progenitor cells on amniotic membrane. Exp Eye Res,2002,74(4):537-45
41 Ohno-M, atsui K, Ichinose S,et al. The effects of amniotic membrane on retinal pigment epithelial cell differentiation. Mol Vis,2005,11:1-10
42 Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology,2001,108(9):1569-74
43闫国和,栗永萍,艾国平,等.人羊膜负载猪角脘细胞重建表皮的形态学研究.第三军医大学学报,2002,24(8):933-6
44闫国和,栗永萍,艾国平,等.人羊膜负载猪角骨髓间充质干细胞生长的形态学研究.第三军医大学学报,2002,24(7):775-7
45 Kallenbach K, Fernandez IIA, Seghezzi G, et al. A quantitative in vitro model of smooth muscle cell migration through the arterial wall using the human amniotic membrane. A rterioscler Thromb Vasc Biol,2003,23(6):1008-13
46 Tryphonopoulos P, Espana EM, Tseng SC, et al. Rat enterocyte cultures on human amniotic membranes. Transplant Proc,2004,36(2):373-4
47 Zhao LP, Chan RMW. Quantitative analysis of neural growth on various substrata especially the human amniotic basement membrane by Eliserrender. Acta Anatomica Sinica,1989,20:71-6
48 Grauss RW, Hazekamp MG, Oppenhuizen F, et al. Histological evaluation of decellularised porcine aortic valves: matrix changes due to different decellularisation methods. Eur J Cardiothorac Surg, 2005,27(4):566-71
49 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983,65(1-2):55-63
1 Yoo JJ, Oberpenning F, Atala A. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology,1998,51(2):221-5
2 Chung SY, Krivorov NP, Rausei V, et al. Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. J Urol 2005, 174(1):353-9
3 Zhang Y, Lin HK, Frimberger D, et al. Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. BJU Int 2005,96(7):1120-5
4 Oberpenning F, Meng J, Yoo JJ and Atala A: De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999,17(2):149-55
5 Atala A, Bauer SB, Soker S, et al. Tissueengineered autologous bladders for patients needing cystoplasty. Lancet 2006,367(9518): 1241-6
6 Zhang Y, Frimberger D, Cheng EY, et al. Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model. BJU Int 2006,98(5):1100-5
7 Badylak SF, Kropp B, McPherson T, et al. Small intestinal submucosa: a rapidly resorbed bioscaffold for augmentation cystoplasty in a dog model. Tissue Eng 1998,4(4):379-87
8 Kropp BP, Rippy MK, Badylak SF, et al. Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J Urol 1996,155(6):2098-104
9 Folkman J, Hochberg MM. Self regulation of growth in three dimensions. J Exp Med,1973,138(4):745-53
10 Atala A, Vacanti JP, Peters CA, et al. Formation of urothelial structures invivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. J Urol,1992,148(2 Pt 2):658-62
11 AtalaA. Bladder regeneration by tissue engineering. BJUInt,2001,88(7):765-70
12 YooJJ,AtalaA. Tissue engineering of genitourinary organs. Ernst Schering Res Found Workshop,2002,(35):105-27
13 Brown AL, Tamara T, Brook-Allred, et al. Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle–urothelial cell interactions. Biomaterials,2005,26(5):529-43
14 Kim BS, Putnam AJ, Kulik TJ. Optimizing seeding and culture methods to engineering smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng,1998,57(1):46-54
15 Qiu QQ,Ducheyne P,Ayyaswamy PS. 3D bone tissue engineered with bioactive microsphers in simulated microgravity. In Vitro Cell Dev Biol Anim,2001,37(3):157-65
16 Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials,2006,27(19):3675-83
17 Liang HC, Chang Y, Hsu CK, et al. Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern. Biomaterials,2004,25(17):3541-52
18 Munting E, Wilmart JF, Wijne A, et al. Effect of sterilization on osteoinduction. Comparison of five methods in demineralized rat bone. Acta Orthop Scand,1988,59(1):34-8
19 Thomas W, Gilbert, Ann M, et al. A quantitative method for evaluating the degradation of biologic scaffold materials. Biomaterials,2007,28(2): 147-150
20 Record RD, Hillegonds D, Simmons C, et al. In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair. Biomaterials,2001,22(19):2653-9
21 Gilbert TW, Stewart-Akers AM, Simmons-Byrd A, et al. Degradation and remodeling of small intestinal submucosa in Achilles tendon repair. J BoneJoint Surg (US),2007,89(3),621-30
22 Rebel JM, de Boer WI, Thijssen CD, et al. An in vitro model of intra-epithelial expansion of transformed urothelial cells. Int J Cancer,1993,54(5):846-50
23 Sutherland RS, Baskin LS, Hayward SW, et al. Regeneration of bladder urothelium, smooth muscle, blood vessels and nerves into an acellular tissue matrix. J Urol,1996,156(2 Pt 2):571-7
24 Mcdevitt CA, Wildey GM, Cutrone RM. Transforming growth factor- beta1 in a sterilized tissue derived from the pig small intestine submucosa. J Biomed Mater Res A,2003,67(2):637-40
25 Hurst RE, Bonner RB. Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence microscopy. J Biomater Sci Polym Ed,2001,12(11):1267-79
26 Lu SH, Sacks MS, Chung SY, et al. Biaxial mechanical properties of muscle-derived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials,2005,26(4):443-9
27 Perchota HJ , Gleason CA ,Dahms SE, et al. Bladder acellular matrix graft: in vivo functional properties of the regenerated rat bladder. Urol Res,1999,27(3):206-13
28 Nuininga JE, van Moerkerk H, Hanssen A, et al. A rabbit model to tissue engineer the bladder. Biomaterials,2004,25(9):1657-61
1 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-7
2 Wakitani S, Saito T, Caplan AT. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.Muscle Nerve,1995,18(12):1417-22
3 Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. Clin Invest,1999,103(5):697-705
4 Fukuda F. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artifograns,2001,25(3):187-93
5孙丽莉,谭玉珍,王海杰,等.骨髓间充质干细胞的克隆培养及其向心肌细胞的诱导分化.复旦学报(医学版),2003,30(6):519-22
6 Liu Y, Song J, Liu W,et al. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyo-genic differentiation. Cardiovasc Res,2003,58(2):460-8
7 Ye NS, Chen J, Luo GA, et al. Proteomic profiling of rat bone marrow mesenchymal stem cells induced by 5-azacytidine. Stem Cells Dev,2006,159(4):665-76
8 Ye NS, Zhang RL, Zhao YF, et al. Effect of 5-azacytidine on the protein expression of porcine bone marrow mesenchymal stem cells in vitro[J]. Genom Proteom Bioinform,2006,4(1):18-25
9 Tomita S, Mickle DA, Weisel RD,et al. Improved heart function with myogenesis and Angiogenesis after autologous porcine bone marrow stromal cell transplantation. Thorac Cardiovasc Surg,2002,123(6):1132-40
10 Tomita S,Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improve damaged heart function.Circulation,1999,100[Supp19]:247-56
11 Xu W, Zhang X, Qian H, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. ExpBiol Med(Maywood),2004,229(7):623-31
12 Rangappa S, Entwistle JW, Wechsler AS, et al. Cardiomyocyte mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. Thorac Cardiovasc Surg,2003,126(1):124-32
13 Shim WS, Jiang S, Wong P, et al. Exvivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells. Biochem BiophysResCommun,2004,324(2):481-8
14 Li H, Yu B, Zhang Y, et al. Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem BiophysResCommun,2006,341(2):320-5
15 Shake JG, Gruber JP, Baumgartner WA, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model:Engraftment and functional affects.Ann Thorae Surg 2002,73(6):1919-26
16 Bayes-Genis A, Roura S, Soler-Botija C, et al. Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cell. Transplant Proc,2005,37(8):4077-9
17 Zhang FB,LI L,Fang B,et al. Passage-restricted differentiation Potential of mesenchymal stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun,2005,336(3):784-92
18 Liu Y, Song J, Liu W, et al. Growth and differentiation of rat bone marrow stromal cells:Does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003,58:460-8
19 Ninomiya K, Takahashi A, Fujioka Y, et al. Transforming growth factor-beta signaling enhances transdifferentiation of macrophages into smooth muscle-like cells. Hyertens Res,2006,29:269-76
20 Alexandrina B, Ana-Maria R, Horia M,et al. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells. European Journal of Cell Biology,2008,78(3):173-84
21 Narita Y, Yamawaki A, Kagami H, et al. Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle celllineage.Cell Tissue Res,2008,333(3):449-59
22 Yoon BS, Yoo SJ, Lee JE, et al. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. Differentiation,2006,74:149-59
23 Wu YC, Cui L, Li G, et al. PDGF-BB initiates vascular smooth muscle-like phenotype differentiation of human bone marrow mesenchymal stem cells in vitro. Zhonghua Zheng Xing Wai Ke Za Zhi,2007,23(4):335-9
24孟寒,阮秋蓉,瞿智玲,等.小鼠骨髓间充质干细胞体外诱导向平滑肌样细胞分化过程中myocardin的表达.中国动脉硬化杂志,2008,16(1):13-16
25张金明,何涛,黄红军.骨髓间充质干细胞体外诱导分化为平滑肌细胞的实验.中国临床康复,2006,10(1):23-26
26 Brown LF, Detmar M, Tognazzi K, et al. Uterine smooth cells express functional receptors (flt-1 and KDR)for vascular permeability factor vascular endothelial growth factor. Lab Invest,1997,76(2):245-55
27 Liu X,Lin CS, Graziottin T,et al. Vascular endothelial growth factor promotes proliferation and migration of cavernous smooth muscle cells.Urol,2001,166(1):354-60
28 Pueyo ME, Chen Y, DAngelo G, et al. Regulation of vascular endothelial growth factor expression by cAMP in rat aortic smooth muscle cells. Exp Cell Res,1998,238(2):354-8
29苗莉,何国祥,景涛等.观察大鼠血管平滑肌细胞与间充质干细胞直接接触培养对细胞的诱导分化.第三军医大学学报,2007,29(9):814-6
30徐正云,马爱群,王亭忠,等.细胞间接触诱导大鼠骨髓间充质干细胞分化为平滑肌细胞.中国心血管杂志,2005,(10)6:416-9
31韩雅玲,康建,李少华.成年鼠骨髓间充质细胞在体外培养中分化为平滑肌细胞.中华医学杂志,2003,(83)9:778-81
32 Song YS, Lee HJ, Park IH, et al. Potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells. Int J Impot Res,2007,19(4):378-85
33 Kobayashia N,Yasu T,Ueba H,et al. Mechanical stress promotes theexpression of smooth muscle-like properties in marrow stromal cells. Exp Hematol,2004,32(12):1238-45
34 Park JS,Chu JSF,Cheng C,et al. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnol Bioemg,2004,88(3):359-67
35 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-7
36陈晓东,董海,周之德.骨髓间充质干细胞体外转化为骨骼肌细胞的初步观察.中华手外科杂志,2004,20(4):230-2
37 Lattanzi L, Salvatori G, Coletta M, et al. High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. Clin invest,1998,101(10):2119-28
38 Edmondson DG, Olson EN. Helix-loop-helix proteins as regulators of muscle-specific transcription. Biol Chem.,1993,268(22):755-8
39张勇,邹仲敏,郭朝华,等. MyoD基因诱导骨髓间充质干细胞分化为成肌细胞的实验研究.第三军医大学学报,2002,24(12):1423-6
40孙占胜,陈振强.成肌分化因子和5-氮杂胞苷联合诱导骨髓间充质干细胞向骨骼肌细胞分化的实验研究.中国修复重建外科杂志,2007,21(12):1371-5
41曾缨,王凌云.骨髓间质干细胞诱导为肌样细胞分化相关基因的表达.中国病理生理杂志,2006,22(9):1665-8
42 Sun Z, Chen Z, Wang B. In vitro differentiation of rat mesenchymal stem cells into skeletal muscle cells induced by myoblast differentiation factor and 5-azacytidine.Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi,2007,21(12):1371-5
43 Rogers JJ,Young HE,Adkison LR,et al. Differentiation factors induce expression of muscle、fat、cartilage and bone in a clone of mouse pluripotent mesenchymal stem cells. Am Surg,1995,61(3):231-6
44 Stocum DL. Development. A trail of transdifferentiation. Science,2002,298(5600):1901-3
45 Chan J,O’Donoqhue K, Gavina M, et al. Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration.Stem Cells,2006,24(8):1879-91
1 Stock U A, Vacanti J P. Tissue engineering: current state and prospects. Annu RevMed,2001,52:443-51
2 Allman A J, Mcpherson T B, Badylak S F, et al. Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response.Transplantation,2001,71(11):1631-40
3 Badylak S, Kokini K, Tullius B,et al. Morphologic study of small intestinal sub mucosa as a body wall repair device. Surg Res,2002,103(2):190-202
4 Kropp BP, Eppley BL, Prevel CD, et al. Experimental assessment of small intestinal submucosa as a bladder wall substitute. Urol,1995,46(3):396-400
5 Kropp BP, Rippy MK, Badylak SF, et al. Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. Urol,1996,155(6):2098-104
6 Record RD, Hillegonds D, Simmons C, et al. In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair. Biomaterials,2001,22(19):2653-9
7 Nuininga J E, Moerkerk H v, Hanssen A, et al. A rabbit model to tissue engineer the bladder. Biomaterials,2004,25(9):1657-61
8 Schultheiss D, Gabouev A I, Cebotari S, et al. Biological vascularizedmatrix for bladder tissue engineering. matrix preparation, reseeding technique and short-term implantation in a porcine model. Urol,2005,173(1):276-80
9 Campodonico R, Benelli A, Michelazzi E, et al. Bladder cell culture on small intestinal submucosa as bioscaffold:experimental study on engineered urothelial grafts. Eur Urol,2004,46(4):531-7
10 Dhams SE, Piechota HJ, Dahiya R, et al. Composition and biome chemical properties of the bladder acellular matrix graft: comparative analysis in rat、pig and human. Br J Urol,1998,82(3):411-9
11 Probst M, Dahiya R, Carrier S, et al. Reproduction of functional smooth muscle tissue and partial bladder replacement. Br J Urol,1997,79(4):505-15
12 Atala A. Technology insight: Applications of tissue engineering and biological substitutes in urology. Nat Clin Pract Urol, 2005,2(3):143-9
13 Wefer J, Sievert K D, Schlote N, et al. Time dependent smooth muscle regeneration and maturation in a bladder acellular matrix graft: Histological studies and in vivo functional evaluation. Urol,2001,165(5):1755-9
14 Reddy P P, Barrieras D J, Wilson G, et al. Regeneration of functional bladder substitutes using large segment acellular matrix allografts in a porcinemodel.Urol,2000,164(3Pt2):936-41
15 Yoo JJ, Meng J, Oberpenning F, et al. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology,1998,51(2):221-5
16 Brown A L, Fathat W, Merguerian P A, et al. 22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model.Biomaterials,2002,23(10):2179-90
17韩平,宋超,魏强,等.组织工程膀胱细胞外基质生物相容性的实验研究.四川大学学报,2007,38(6):1009-12
18 Hattori K, Joraku A, Miyagawa T ,et al. Bladder reconstruction using a collagen patch prefabricated within the omentum. Int J Urol,2006,13(5):529-37.
19杨中华,王行环,王怀鹏,等.以纤维蛋白凝胶为支架构建组织工程胃新膀胱的研究.现代泌尿外科杂志,2007, 12(5):305-7
20 Atala A, Bauer S B, Soker S, et al. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet,2006,367:1241-6
21 Chung S Y. Bladder tissue-engineering: a new practical solution? Lancet,2006,367:1215-6
22 Shinoka T, Shum-Tim D, Ma PX, et al. Creation of viable pulmonary artery autografts through tissue engineering. Thorac Cardiovasc Surg,1998,115(3):536-46
23 Scheepens WA, Debie RA, Weil EH, et al. Unilateral versus bilateral sacral neuromodulation in patients with chronic voiding dysfunction.Urol,2002,168(5):2046-50
24 Cook AD, Hrkach JS, Gao NN, et al. Characterization and development of RGD-peptide-modied? poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial. J Biomed Mater Res, 1997,35:513-23
25 Barrera DA, Zylstra E, Lansbury PT, et al. Co-polymerization and degradation of poly(lactic acid-co-lysine). Macromolecules,1995, 28:425-32
26 Oberpenning F, Meng J, Yoo JJ, et al. De novo reconstruction of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol,1999,17:149-55
27 Atala A. Tissue engineering for bladder substitution. World J Urol,2000,18(5):364-70
28 Kirk D, Kagawa S, Vener G, et al. Selective growth of normal adult human urothelial cells in serum-free medium. In Vitro Cell Dev Biol 1985,21(3pt1):165-71
29 Cilento BG, Freeman MR, Schneck FX, et al. Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro.Urol,1994,152:655-70
30杨中华,王行环,杨梅超,等.猪膀胱移行上皮细胞培养及其在纤维蛋白凝胶表面生长的评价.中国临床康复,2006,10(17),61-4
31 Baskin LS , Howard PS, Duckett JW, et al. Bladder smooth muscle cells in culture: identification and charactarization. J Urol,1993,149:190-7
32 Sui GP, Wu C, Fry CH. The electrophysiological properties of cultured and freshly isolated detrusor smooth muscle cells. J Urol,2001, 165:627-33
33卢慕峻,王忠,傅强,等.组织工程化膀胱平滑肌结构的体外构建.组织工程与重建外科杂志,2006,2(2),76-8
34 Chung SY, Krivorov NP, Rausei V, et al. Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. Urol,2005,174(1):353-9
35 Schultheiss D, Gabouev A, Pilatz A, et al. Use of bone marrow derived mesenchymal stem cells for autologous tissue engineering of urinary bladder muscularis. European Urology Supplements, 2004, 3:159
36 Frimberger D, Morales N, Gearhart J, et al. Human embryoid body-derived stem cells in tissue engineering—enhanced migration in co-culture with bladder smooth muscle and urothelium.Urol,2006,67(6),1298-303
37 Lakshmanan Y ,Frimberger D ,Gearhart J, et al. Human embryoid body-derived stem cells in co-culture with bladder smooth muscle and urothelium.Urol,2005,65(4):821–6
38姚康德,宋雪峰,刘文广,等.组织工程研究与开发进展.中国修复重建外科杂志,2002,16(5):325-8
39 Kim BS, Putnam AJ, Kulik TJ. Optimizing seeding and culture methods to engineering smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng,1998,57(1):46-54
40 Qiu QQ,Ducheyne P,Ayyaswamy PS. 3D bone tissue engineered with bioactive microsphers in simulated microgravity. In Vitro Cell Dev Biol Anim,2001,37(3):157-65
41 Fauza Do, Fishman S, Mehegan K, et al. Videofeto-scopically assisted fetal tissue engineering: Bladder augmentation. Ped Surg,1998,33:7-12
42 Zhang Y, Kropp BP,Moore P,et al. Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa:potential applications for tissue engineering technology.Urol,2000,164(3Pt2):928-5
43 Danielsson C, Ruault S, Basset-Dardare A ,et al. Modified collagen fleece,a scaffold for transplantation of human bladder smooth muscle cells.Biomaterials,2006,27(7):1054-60